Capillary electrophoresis using replaceable gels

ABSTRACT

The filling of an internally coated capillary with a gel in its polymerized state without damaging the gel. The coating prevents bonding of the gel to the inside of the capillary, The gel comprises up to 6% acrylamide and 0-5% crosslinker. The gel can be advantageously and conveniently used in automated electrophoresis systems for automatic replacement of spent gel.

This is a division of application Ser. No. 07/793,256, filed Nov. 13,1991, now U.S. Pat. No. 5,332,481, which is a continuation ofapplication Ser. No. 07/647,071, filed Jan. 29, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to capillary gel electrophoresis, and moreparticularly to refilling capillaries using a polymerized gel.

2. Description of Related Art

Electrophoresis is one of the most widely used separation techniques inthe biologically related sciences. Molecular species such as peptides,proteins, and oligonucleotides (analytes) are separated by causing themto migrate at different rates in a separation medium under the influenceof an electric field. The separation medium can be a buffer solution, ora low to moderate concentration of an appropriate gelling agent such asagarose or polyacrylamide. When gel separation medium is used,separation of analytes is partly based on their molecular sizes as theanalytes are sieved by the gel matrix. Smaller molecules move relativelymore quickly than larger ones through a gel of a given pore size whichdepends in part on the concentration of the polymer in the gel.

U.S. Pat. Nos. 4,855,706 and 4,865,707 to Barry L. Karger and AharonsCohen describe gel compositions suitable for capillary electrophoresis.A fused silica capillary having inner diameter in the order of 75 μm isfirst filled with a mixture of acrylamide monomer and other ingredientsand polymerization is then allowed to go to completion in the capillary.The time taken to complete polymerization is a minimum of one hour. Thepolymerized gel has a limited storage life. Also, performance of the geldeteriorates after a period of use. This may be due to gradualaccumulation of macromolecules in the gel matrix after repeated runs.The applied electric field may cause disintegration of the polymermaterial after repeated use. In the past, the gel-filled capillarycolumns have to be discarded after their useful life.

The gel columns have heretofore been used in laboratory set-upsinvolving many manual steps, e.g. placement of buffer containers withrespect to the ends of the gel column, etc. Also spent gel column has tobe manually replaced by a new gel column. To take advantage ofautomation in carrying out electrophoresis, due considerations should begiven to eliminate as many of the manual steps in the design of anautomated electrophoresis system.

SUMMARY OF THE INVENTION

The present invention is directed to filling an internally coatedcapillary using gel which is polymerized before filling the capillary.The internal coating on the capillary walls prevents bonding of the gelto the capillary walls. The gel is of a composition which allows it tofill the capillary in its polymerized state without damage to the geland to be removed from the capillary after the gel has expended itsuseful life. The capillary can then be refilled with fresh gel. This canbe handled automatically by an automated capillary electrophoresissystem.

In the illustrated embodiment, the formulation of the gel comprises upto 6% of acrylamide monomer and buffer. In addition, optional amounts ofcrosslinker, catalyst, initiator, urea, ,and other additives may beadded to adjust for the desired pore size, separation efficiency andlife of the gel. The composition results in a gel of a consistency whichcan be forced into and out of the internally coated capillary withoutdamaging the gel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic layout of an automated electrophoresis system.

FIG. 2 is an electropherogram representing the results ofelectrophoresis using gel-injected into the capillary in polymerizedstate according to the present invention.

FIG. 3 is an electropherogram representing the results ofelectrophoresis using the same gel by which has been polymerized in thecapillary.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best presently contemplated mode ofcarrying out the invention. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in a limiting sense. The scope of the invention is best determinedby reference to the appended claims.

The present invention can be used advantageously in conjunction withautomated capillary electrophoresis systems, such as the P/ACE™ 2000electrophoresis system introduced by Beckman Instruments, Inc. Saidsystem is schematically shown in FIG. 1. The details of the system havebeen described in copending U.S. patent applications Ser. No.07/614,059; 07/542,673 and 07/187,760 commonly assigned to the assigneeof the present invention and are incorporated by reference-herein. Inthat system, the capillary column 10 is encased in a cartridge 12 whichis supported to allow the ends of the capillary to access vials 14 and16 of electrolyte or sample solutions. Capillary referred herein meanstubing having inside diameter typically less than 1000 μm and moretypically less than 300 μm. A detector 13 is provided to detectseparated species. The vials are carried on carousels 18 and 20 whichare rotated to position selected vials at the ends of the capillary. Aselected solution can be forced into the capillary by submerging one endof the capillary into the vial and pressurizing the vial by means notshown (for details see copending applications). According to the presentinvention, the polymerized gel can be contained in a vial on thecarousel and forced into an internally coated fused silica capillary inthe same manner as is done with solution. The capillary walls are coatedwith a material (e.g. 50% phenyl and 50% methyl, or cynopropyl) toprevent bonding of the gel to the walls. Since the instrument has abuilt-in rinse mode which runs a rinsing solution (typically a buffer orelectrolyte solution) through the capillary to clean the capillary, arinsing solution can thus be used to wash out the spent gel. Thecapillary 10 can then be refilled with fresh gel. Alternative, insteadof using a rinse solution, a supply of fresh gel can be used to displacethe spent gel thus flushing and refilling the capillary in a singlestep. The above steps can be carried out automatically under control ofmicroprocessor 30 programmed by the user. It can be seen that it wouldnot be necessary to replace the entire gel capillary column 10 toreplace the gel which would involve removing cartridge from the systemand removing the column from the cartridge 12.

The P/ACE™ system also has a sample injection mode which injects samplefrom a vial into one end of the capillary by either electromigration orpressure injection. Electrodes 26 and 28 are provided to apply therequired high voltage (in the order of several hundred volts per cm ofcapillary) from voltage supply 29 for electromigration injection as wellas for carrying out electrophoresis. Electrophoresis is performed withthe two ends of the capillary dipped into electrolyte containing vials.The electrolyte can be in the form of buffer solution similar to thebuffer the gel is made up of, or in the form of gel (i.e. a gel buffersystem). In the latter case, the gel in the capillary can be replacedwithout having to position another vial.

The basic composition of the refillable gel is up to 6% acrylamidemonomer dissolved in the appropriate buffer solution (usually 100 mMTRIS-borate of pH about 8.5). The acrylamide can be cross-linked with 0to 5% of methylenebisacrylamide ("BIS"). 7M urea, hydrophilic polymeradditives (e.g. polyethyleneglycol ("PEG")), an appropriate amount ofcatalyst (e.g. tetramethyleneethylenediamine ("TEMED") and initiator(e.g. ammonium persulfate) and other additives may be added to obtain agel having the desired pore size, separation efficiency and life span.The steps for preparing the buffer and the gel are conventional and wellknown to one skilled in the art. Generally, the composition is allowedto polymerize overnight and the polymerized gel can be dialyzed orelectrodialyzed against the gel buffer in order to remove the remainingammonium persulfate, TEMED and the non-polymerized acrylamide andBIS-monomers if necessary. The coated inner surface of the fused silicacapillary can be treated before the first filling of the gel. Thesurface can be treated by using 100% solution ofmethacryloxypropyltrimethoxysilane for 1 hour at 50° C. A dilutedsolution (diluted with methanol) may also be used. The silane is for"neutralizing" any hydroxide ions remaining on the capillary walls as aresult of exposed silica due to slight imperfection in the coating. Thepresence of hydroxide ions is undesirable for some applications as itincreases electroendoosmosis.

The capillary can be refilled with fresh gel of the same or differentcomposition right after the previously spent gel has been removed fromthe capillary. Unlike the prior art gel columns where polymerizationtakes place in the capillary, it will not be necessary to wait forpolymerization to take place in the present invention once the capillaryhas been filled with polymerized gel. This complements the automatedfeatures of the automated electrophoresis system and eliminates waitingtime for changing of gel columns.

Up to 6% acrylamide without crosslinker, or up to 2% acrylamide +5%crosslinker, PEG of molecular weight up to 35,000 at concentration of upto 1% can be used is additive. Gels having high concentrations ofpolyacrylamide and crosslinker are too brittle to be able to be forcedin their polymerized state to fill the capillary without damaging thegel. However, it is believed that gel having acrylamide greater than 6%may also maintain its integrity under special injection conditions.Since prior art gel-filled capillary columns do not have internalcoating which prevents bonding of the gel to the capillary, the spentgel cannot be pushed out of the capillary effectively as the gel bondsto the capillary walls. It is noted that at concentration above 6% ofacrylamide without crosslinker, an appropriate increase of PEGconcentration and molecular weight is necessary to maintain therefillable property of the gel i.e. viscosity. Similarly, it has beenfound that in a composition having 5% BIS monomer and more than 2%acrylamide monomer, the PEG concentration and molecular weight should beincreased.

Examples of specific compositions of the refillable gels according tothe present invention which performance have been found to be comparableto prior art non-refillable gels are given below. The following examplesare offered for illustrative purposes only, and are intended neither todefine nor limit the invention in any manner.

EXAMPLE 1

100 mM TRIS

100 mM Boric Acid

3% T

0.5% C

2 mM EDTA (for separation of polynucleotides)

8.35 pH

EXAMPLE 2

100 mM TRIS

100 mM Boric Acid

1% T

5% C

2 mM EDTA (for separation of polynucleotides)

8.35 pH

In both examples above, the acrylamide and crosslinker concentrationsare expressed in %T and %C to characterize the gels. The definitions of%T and %C are as follows: ##EQU1##

The total amount as well as the ratio of acrylamide and crosslinkerdetermine the pore size and the pore distribution of a polyacrylamidegel. In the examples given, there is 2.985% acrylamide and 0.015%crosslinker (BIS) in Example 1 and 0.95% acrylamide and 0.05%crosslinker in Example 2. The crosslinker may be omitted in Example 1 ifdesired.

The results of electrophoresis using the gel of Example 1 is representedby the electropherogram in FIG. 2. The sample undergoing electrophoresisis φ-X 174 RF DNA-Hae III Digest mixture. The capillary column has aninner coating (e.g. OV-17) for preventing bonding of the gel to thecapillary walls. The dimension of the capillary is 100 μm I.D., 47 cmtotal length with 40 cm effective length. The gel was polymerized andthen injected into the capillary by using the rinse mode of the P/ACE™2000 system. The sample is of 1 mg/ml concentration and is injected byelectromigration into the gel column by applying 5 KV for 2 seconds. Theelectrophoresis is carried out under 12 KV and 33 μA.

The results in FIG. 2 can be compared to the results shown in FIG. 3which represents the electropherogram of the same sample separated in agel column having the same composition but which has the gel polymerizedin the column. Sample injection and run parameters are the same. It isseen that the resolution of the peaks in the two electropherograms arequite similar. While the amplitudes between the two results differsomewhat, it is however not as much a concern as peak resolution forpurposes of gel electrophoresis analysis as it is not a quantitativeanalysis.

Accordingly, it has been demonstrated that gel columns that have beenrefilled according to the present invention provide substantially thesame separation efficiency, power and resolution as compared to the samegel polymerized in the capillary. The gel is not damaged as it is beingforced into the coated capillary in its polymerized state therebymaintaining its separation performance. The refillable gel can be usedadvantageously in automated electrophoresis systems in which the task ofreplacing fresh gel can be handled automatically. A series of runsincluding changing of gel between runs can be programmed to be performedautomatically without user intervention.

While the invention has been described with respect to the preferredembodiments in accordance therewith, it will be apparent to thoseskilled in the art that various modifications and improvements may bemade without departing from the scope and spirit of the invention.Accordingly, it is to be understood that the invention is not to belimited by the specific described embodiments, but only by the scope ofthe appended claims.

I claim:
 1. An apparatus for separating a sample into its molecularspecies comprising:a capillary; a supply of polymerized gel disposed incommunication with one end of the capillary; means for filling thecapillary with the polymerized gel; means for introducing the sampleinto the filled capillary; and means for performing electrophoresis onthe sample to separate the sample into its molecular species.
 2. Asystem as in claim 1 further comprising means for removing spent gelfrom the capillary so that it can be refilled with polymerized gel.
 3. Asystem as in claim 2 further comprising means for automatic control ofoperation of the system.
 4. A system as in claim 1 wherein thepolymerized gel and sample are contained in receptacles and wherein thesystem further comprises a carousel for supporting the receptacles andmeans for turning the carousel to position the carousel to position therespective receptacle in flow communication with the capillary.
 5. Asystem as in claim 4 wherein the means for filling the capillary andintroducing the sample comprises means for pressurizing the receptaclesin order to effect gel filling and sample introducing.
 6. A system as inclaim 5 further comprising means for automatic control of operation ofthe system.
 7. A system as in claim 6 wherein the means for automaticcontrol is programmed to displace spent gel originally in the capillaryby filling polymerized gel from the receptacle into the capillary.
 8. Asystem as in claim 7 further comprising means for analyzingelectrophoretically separated components of the sample.
 9. A system asin claim 8 further comprising a cartridge for supporting the capillary.10. A system as in claim 1 wherein the capillary has inner walls coatedwith a material that prevents bonding of the polymerized gel to thewalls.
 11. A system as in claim 10 wherein the polymerized gel has acomposition comprising up to 6% acrylamide and 0-5% crosslinker.
 12. Agel-containing capillary column useful for electrophoresis comprising:acapillary having a bore coated with a material having a property whichprevents bonding of gel to the material; and a polymeric gel fillingsaid bore, wherein the gel has a composition that allows it to be forcedto fill the bore substantially without damage to gel structure whichmight otherwise significantly affect the electrophoretic performance ofthe gel.
 13. A capillary column as in claim 1 wherein the gelcomprises:up to 6% of acrylamide; and 0-5% of crosslinker.
 14. Acapillary column as in claim 13 wherein the crosslinker comprisesmethylenebisacrylamide.
 15. A capillary column as in claim 14 whereinthe coated material contains either phenyl groups and methyl groups orcyanopropyl groups.
 16. A capillary electrophoresis system comprising:acapillary; a supply of polymerized gel disposed in communication withone end of the capillary; a supply of sample; means for filling thecapillary with the gel and introducing a sample into the capillary; andmeans for performing electrophoresis.
 17. A system as in claim 16further comprising means for removing the gel from the capillary so thatit can be refilled with gel.
 18. A system as in claim 17 furthercomprising means for automatic control of the operation of the system.19. A system as in claim 26 wherein the gel and sample are contained inreceptacles and wherein the system further comprises a carousel forsupporting the receptacles and means for turning the carousel toposition the carousel to position the respective receptacle in flowcommunication with the capillary.
 20. A system as in claim 19 whereinthe means for for filling the capillary and introducing the samplecomprises means for pressurizing the receptacles in order to effect gelfilling and sample introducing.
 21. A system as in claim 20 furthercomprising means for automatic control of the operation of the system.22. A system as in claim 21 wherein the means for automatic control isprogrammed to displace gel originally in the capillary by filling gelfrom the receptacle into the capillary.
 23. A system as in claim 22further comprising means for analyzing electrophoretically separatedcomponents of the sample.
 24. A system as in claim 23 further comprisinga cartridge for supporting the capillary.
 25. A system as-in claim 16wherein the capillary has inner walls coated with a material thatprevents bonding of the gel to the walls.
 26. A system as in claim 25wherein the gel has a composition comprising up to 6% acrylamide and0-5% crosslinker.